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Diagnostic Test HIV Resistance Test

Diagnostic Test HIV Resistance Test. Thumbi Ndung'u, BVM, PhD. Nelson R. Mandela School of Medicine. Evolution of Viral Mutations. Mutations arise because HIV-1 RT makes spontaneous errors (1 in 10 4 )

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Diagnostic Test HIV Resistance Test

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  1. Diagnostic TestHIV Resistance Test Thumbi Ndung'u, BVM, PhD. Nelson R. Mandela School of Medicine

  2. Evolution of Viral Mutations • Mutations arise because HIV-1 RT makes spontaneous errors (1 in 104) • HIV-1 genome is 10,000 (104) bases long, therefore 1 error each time the genome is replicated • Production of virus = 109 to 1010virions per day  quasispecies • Every possible mutation present in quasispecies before ARV therapy

  3. Selection of Resistant strains Treatment begins Drug-susceptible quasispecies Drug-resistant quasispecies Selection of resistant quasispecies Viral load • Incomplete suppression • Inadequate potency • Inadequate drug levels • Inadequate adherence • Pre-existing resistance Time

  4. Mutations(cont) • Mutations described in ff way: e.g. M184V • Initial letter represents the wild-type amino acid • Number represents the mutated codon • End letter represents the mutant amino acid

  5. 1.PIs Primary mutations: V32I, G48V, I50V, V82A/F/T/S, I84V and L90M. Secondary/accessory mutations: 46, 47, 53, and 54 Polymorphisms associated with resistance: 10, 20, 36, 63, 71, 77, and 93 Patterns of resistance

  6. Mutations associated with resistance to PIs

  7. Patterns of resistance (cont) Selected by AZT and/or d4T Accumulation of mutations 41L, 67N, 70R, 210W, 215Y/F, 219Q/E 3TC M184V 2.NRTIs • Thymidine Analog Mutations (TAMS) The OH group (essential for continuation of the growing chain) is absent in AZT and d4T

  8. Effect of TAMS and M184V • Cross-resistance with d4T, ddI, ddC, 3TC • 2 TAMS + M184V significantly reduces potency of ABC • 3 TAMs including M41L or L210W significantly reduces activity of TDF • M184V (3TC mutation) reverses the effect of the T215Y/F • but, M184V effect is lost with multiple TAMS

  9. 3.NNRTIs NVP (Nevirapine) L100I, K103N, V106A/M, V108I, Y181C/I, Y188C/L/H, G190A EFV (Efavirenz) L100I, K103N, V106M, V108I, Y181C/I, Y188L, G190A/S, G225H Patterns of resistance (cont)

  10. How do you measure drug resistance? Genotyping: Indirect assay: Detects drug resistance mutations that are present in the relevant virus genes. Phenotyping: Direct assay: Measures the ability of the virus to grow in various concentrations of antiretroviral drugs.

  11. F B A H G C 1.8kb Genotyping using the Viroseq Kit plasma dried blood spot protease RT Load samples onto ABI 3100

  12. Sequence Analysis

  13. http://hivdb.stanford.edu/

  14. Final Diagnosis Multidrug resistant HIV

  15. References http://hivdb.stanford.edu http://www.hivresistanceweb.com http://hiv.medscape.com/Home/Topics/AIDS/AIDS.html http://www.hivatis.org http://hiv-lanl.gov/seq-db.html

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